ABSTRACT Acinetobacter baumannii is listed by the CDC as a clinical pathogen that poses a serious antibiotic resistance threat in the United States. A. baumannii is resistant to antimicrobial agents of different classes, but the most troublesome is resistance to the last resort carbapenem antibiotics, which were the drugs of choice for treatment of infections caused by this microorganism. The major mechanism of resistance of A. baumannii to carbapenems is production of antibiotic-inactivating enzymes, carbapenem-hydrolyzing class D ?-lactamases or CHDLs. In addition, sensitivity of carbapenem targets, bacterial penicillin-binding proteins (PBPs), rates of antibiotic penetration into the bacterial cell and/or their expulsion by efflux pumps can also contribute to resistance. Levels of resistance to carbapenem antibiotics reach up to 90% in some parts of the world, and mortality rates from infections caused by such bacteria are staggeringly high, up to 50%. Our long-term goal is to develop novel antibiotics for treatment of deadly A. baumannii infections. During the first cycle of funding for this grant proposal, we performed in-depth characterization of clinically important CHDLs and proposed the mechanism for their carbapenemase activity, which provides guidance for development of a new generation of carbapenems capable of inhibiting these enzymes. The continuation of this research will be a collaborative effort with Dr. John Buynak (co-PI) who developed dozens of novel atypically- modified carbapenem antibiotics. We used these antibiotics to test our proposed mechanism for deacylation and found three that inhibit the most prevalent A. baumannii CHDL, OXA-23, and possess superior activity (when compared to commercial carbapenems) against OXA-23-producing A. baumannii. Our proposed studies are aimed at in-depth characterization of these promising novel drugs. We will determine activity of our compounds against A. baumannii strains expressing major CHDLs and unveil kinetic and structural features responsible for their ability to inhibit these enzymes (Aim 1). We will evaluate interaction of our novel carbapenems with their targets, PBPs, and determine to what extent efflux pumps and porins influence bacterial resistance to these antibiotics (Aim 2). Finally, we will design and characterize several dozen novel carbapenem antibiotics with the aim to further improve their antimicrobial activity by enhancing their inhibitory potency against CHDLs, improving affinity for PBPs and increasing penetration rates and resistance to efflux (Aim 3).